The project represents a continued study of the biochemical cytology of Trichomonas vaginalis, a sexually transmitted common parasite that causes vaginitis and urethritis in humans. Based on recent results the scope of the project is extended to include several other protists parasitic in humans (and also livestock): other trichomonad species, Giardia lamblia, Entamoeba histolytica, rumen ciliates and a rumen fungus. These organisms all share a number of basic metabolic properties of which three need to be stressed: they have a fundamentally 02 independent energy metabolism, lack mitochondria and possess a pyruvate decarboxylating enzyme, pyruvate:ferredoxin oxidoreductase that is fundamentally different from the corresponding mitochondrial enzyme, pyruvate dehydrogenase complex. Trichomonads and the rumen protists, but not G. lamblia or E. histolytica, contain hydrogenosomes, organelles of pyruvate oxidation leading to hydrogen formation. The project aims at furthering our understanding of the nature and evolutionary origin of the anaerobic mode of life in the organisms studied. Special attention is devoted to the hydrogenosome and its biological affinities. Work in the next project period will focus on the establishment of the primary structure of a set of characteristic enzymes of anaerobic metabolism of the organisms in question. Work on T. vaginalis enzymes is primarily based on partial (amino-terminal and internal) sequencing of purified proteins, polymerase chain reaction (PCR) mediated amplification of cDNA followed by sequencing with the use of oligonucleotide probes designed from the amino acid sequences. The same approach will also be used for those other organisms that are easily cultured. Organisms not amenable to cultivation will be studied by developing degenerate oligonucleotide probes for PCR based on sequences conserved over a large phylogenetic span in isofunctional enzymes. Sequences obtained will be compared with data on the other protists studied and with sequences in the databases. Degrees of similarities will be used to establish evolutionary relationships of the enzymes studied and these relationships will be correlated with information of the evolutionary relationships of the organisms studied.